Methionine-Decorated Near Infrared Fluorescent Probe for Prolonged

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Methionine Decorated Near infrared fluorescent probe for Prolonged Tumor Imaging Chen Wei, Zhenwei Yuan, Jinrong Zheng, Habtamu Kassaye, Lijuan Gui, Fei Wang, Hao Wan, Yue Xu, Qing He, Murat Er, Yi Ma, and Haiyan Chen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00233 • Publication Date (Web): 13 Jun 2018 Downloaded from http://pubs.acs.org on June 14, 2018

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Molecular Pharmaceutics

Methionine Decorated Near infrared fluorescent probe for Prolonged Tumor Imaging Chen Wei, Zhenwei Yuan, Jinrong Zheng, Habtamu Kassaye, Lijuan Gui, Fei Wang, Hao Wan, Yue Xu, Qing He, Murat Er, Yi Ma *, Haiyan Chen * Department of Biomedical Engineering, School of Engineering, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Lane, Gulou District, Nanjing 210009, China

*Author to whom correspondence should be addressed: Yi Ma Email:[email protected] Haiyan Chen, PhD Email: [email protected] Tel: +86-25-83271080 Fax: +86-25-83271046

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Methionine (Met), one of the essential amino acids, of which the transport system L is over-expressed in various tumor cells. In this study, a near-infrared fluorescent dye (IR-780) and methionine were conjugated through a piperazin-polyamines linker to form Cy-Met. The successful synthesis of Cy-Met was validated by optical characterization, NMR and MS spectra. The absorption peak of Cy-Met was at 680 nm and fluorescence peak was at 790 nm. Cytotoxicity assay and cell imaging studies indicated that Cy-Met had good biocompatibility and specific affinity to tumor cells. The dynamic distribution and clearance investigations showed that Cy-Met was eliminated by the liver-intestine pathway. Notably, Cy-Met displayed tumor-targeting ability in U87, H22 and EAC tumor-bearing mice with an evident long circulation time. The results implied that Cy-Met could act as a promising fluorescence probe specialized for long-term tumor monitoring. Key words: Methionine, targeting, fluorescent probe, tumor imaging, long circulation


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1. Introduction The development of effective detecting method with more sensitivity and specificity for the visualization of tumors is highly needed and is crucial to improve the current clinical treatment of tumors.1-3 To date, fluorescent probes are of great importance in diagnosis and imaging of many diseases, such as inflammation, coronary heart, hypertension and cancers.4-6 Notably, near-infrared (NIR) fluorescence probes combined with NIR fluorescence imaging system are characterized with convenience, high penetration, non-invasiveness and non-radioactivity especially due to the reduction of strong intrinsic auto-fluorescence background from living tissues and avoiding the interference from biological systems.7,8 Currently, the strategy for constructing active tumor-targeting fluorescent probes is conjugating of probes with appropriate tumor-targeting ligands, such as proteins, peptides (RGD), antibodies and glucose derivative (Hyaluronic acid) and so on.9-11 Ye et al, synthesized and evaluated a series of multimeric RGD compounds to link to a dicarboxylic acid-containing NIR fluorescent dye (cypate) for tumor targeting.12 Deng et al. synthesized a novel pH and reduction dual-sensitive hyaluronic acid mineralized micelle for efficient intracellular drug delivery.13 Mahalingam et al, described the use of a 10 kDa protein scaffold, a Centyrin, conjugated with a NIR fluorescent dye to diagnose tumors that overexpress the EGFR.14 König et al, developed diverse NIR-emissive cyanine dyes to conjugate with folic acid for cancer cell staining.15 Koner et al, synthesized the first substrate mimicking fluorescent probe which could differentiate folate-receptor-positive and negative cells successfully.16 However, these ligands are limited by only targeting to partial types of cancer cells. Hence, developing a new tumor-targeting fluorescent probes those are suitable to most of the tumor cells is particularly important for tumor diagnosis and monitoring after tumor therapy. It was reported that endogenous amino acids could be used as functional moiety for tumor-targeting.17 System L, as the most common transport system involved in amino acids transportation, includes LAT1, LAT2, LAT3, and LAT4, which are responsible for transporting L-amino acids.18 Many studies have shown that LAT1 is consistently ACS Paragon Plus Environment


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expressed at high levels in cancer cells in comparison with normal cells. Many derivatives of L-amino acids, including various halogenated derivatives of tyrosine, phenylalanine and methylaminoisobutyric acid (MeAIB) have also been proved to enter the cells using similar transport systems. Liu et al. showed that L-lysine labeled with [99mTc(CO)3]+ could still be recognized by the LAT1.19 Other essential amino acids (such as methionine, tyrosine, glycine, phenylalanine and leucine) and their analogues have been explored as tumor-target agents for fluorescence imaging.20 Pauleit

et

al,

synthesized

a

very

18

promising

F-labeled

amino

acid,

O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET), which exhibited high uptake in tumor cells.21 Fisher et al, developed a tumor-targeting inhibitors through combining corynebacterium parvum with L-phenylalanine mustard or methotrexate.22 Methionine (Met), an essential amino acid, was substituted for by its precursor homocysteine (Hcy) in the culture medium.23,24 “Methionine dependency”, one of the metabolic characters of cancer cells, occurs in various human cancer cells including breast, colon, lung, kidney, bladder, neuroglia and melanin cells.25,26 As Breillout and Kreis pointed out, the higher the metastatic potential of the cell line, the higher the concentration of Met required to maintain its proliferation.27,28 Herein, a novel Met conjugated fluorescent probe (Cy-Met) based on NIR fluorophore (IR-780) for tumor imaging was designed and synthesized. It exhibited a strong emission at 790 nm, which was qualified for penetration through the deep tissues. Cy-Met also demonstrated good biocompatibility in tumor cells. Furthermore, the in vivo tumor-targeted imaging by Cy-Met were evaluated on tumor-bearing mice models. Along with the elongation of time, the fluorescence signal shown in tumor site lasted for a long time up to a few days. The results revealed a promising application of this NIR fluorescent probe for tumor imaging. 2. Experimental Section 2.1 Reagent and Instruments N-(3-Bromopropyl)

Phthalimide,

L-methionine

and

2-amino-bicyclo-(2,2,1)

heptane-2-carboxylic acid (BCH) were purchased from Macklin Biochemical Co. Ltd (Shanghai, China). Hoechst 33342 and Mito-Tracker Green were purchased from ACS Paragon Plus Environment

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KeyGen

BioTech

(Jiangsu,

China).

3-(4,

5-Dimethylthialzol-2-yl)-2,

5-diphe-nyltetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin-EDTA were purchased from the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). All other chemicals were supplied by J&K Scientific Ltd. (Beijing, China) or Sinopharm chemical reagent Co. Ltd. (Shanghai, China) and utilized without further purification. Ultrapure water from a Milli-Q reference system (Millipore) was utilized in all experiments. Phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH=7.4) solution was purchased from Invitrogen. The compounds were characterized by 1H NMR, 1

13

C NMR, and ESI-MS spectra.

H NMR spectra was detected by Bruker 600 MHz and

13

C NMR spectra was

detected by Bruker 151 MHz spectrometer, in which tetramethylsilane (TMS) was utilized as internal standard (0 ppm) substances, and DMSO-d6/CDCl3 was used as solvent. Mass spectra were collected on tandem quadrupole mass spectrometer (Waters, Milford, MA, USA) with ESI resource and AB SCIEX 5800 matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (SCIEX, Beijing, China) respectively. 2.2 Synthesis of Cy-Met PyBop (40 mg, 0.071 mmol) and N, N-diisopropylethylamine (DIEA) (9.17 mg, 0.07 mmol) were added to a solution of L-methionine (10.6 mg, 0.071 mmol) in anhydrous DMF (2 mL) under a nitrogen atmosphere was added. After the resulting solution was stirred for 30 min at 30 °C, a solution of Cy-NH2 (50 mg, 0.064 mmol) in anhydrous DMF (1 mL) was added dropwise at room temperature in nitrogen protected environment.29-33 The reaction was monitored by thin-layer chromatography (TLC). The solvent was removed under reduced pressure after further stirring for 2 h at room temperature. The resulting residue was diluted with dichloromethane (10 mL) and washed with H2O (2 × 5mL) for thrice. The organic layer was dried over Na2SO4, filtered and concentrated in vacuum. The product was purified by silica gel column chromatography with dichloromethane/methanol (20:1) as the eluent to acquire a blue purple solid powder: yield 38.1 mg, 63%. 1H NMR (600 MHz, Chloroform-d) δ: 8.41 ACS Paragon Plus Environment


(d, J = 7.6 Hz, 2H), 8.00 (t, J = 7.6 Hz, 2H), 7.91 (dd, J = 14.9, 7.7 Hz, 4H), 7.33 – 7.27 (m, 3H), 7.19 – 7.07 (m, 2H), 7.05 – 6.92 (m, 2H), 3.81 – 3.71 (m, 3H), 3.24 – 3.18 (m, 2H), 2.50 – 2.41 (m, 4H), 1.98 (t, J = 6.1 Hz, 24H), 1.73 – 1.71 (m, 2H), 1.67 (d, J = 4.9 Hz, 3H), 1.57 (d, J = 7.4 Hz, 2H), 1.54 (d, J = 6.7 Hz, 4H), 1.46 (d, J = 6.6 Hz, 4H), 1.07 – 0.97 (m, 6H). 13C NMR (151 MHz, Chloroform-d) δ: 159.76 , 134.98 , 133.72 , 129.38 , 127.55 , 123.41 , 54.99 , 48.14 , 43.13 , 28.82 , 26.27 , 25.29 , 20.57 , 18.63 , 17.23 , 12.42 , 11.68. HRMS (ESI, m/z): calculated for C48H69N6OS [M]+, 777.5248, found: 777.5242. 2.3 Optical Properties Characterization Absorption spectra were recorded on Hitachi U-3310 spectrophotometer (Hitachi Tokyo, Japan), and fluorescence spectra were obtained by using PerkinElmer-LS55 spectrophotometer (PerkinElmer, USA). All UV-Vis and fluorescence spectra measurements were carried out in PBS solution containing 1 % DMSO, pH 7.4. Absorption spectra were collected in the range from 450 nm to 900 nm. Fluorescence spectra were recorded in the range from 700 to 950 nm withλex =680 nm. 2.4 Cytotoxicity Assay experiments MCF-7 cell (Human breast cancer cell), A549 cell (Adenocarcinomic human alveolar basal epithelial cell), U87 cell (Human Glioblastoma cell), L02 cell (Normal human hepatic cell) and HUVEC cell (Human umbilical vein endothelial cell) were obtained from American Type Culture Collection (ATCC, USA). The cell lines were cultivated on glass-bottom culture dishes with an atmosphere of 5 % CO2 at 37 °C in DMEM supplemented with 10 % FBS and 1 % (v/v) penicillin-streptomycin. MTT assay was used to investigate the cytotoxicity of Cy-Met on L02 and U87 cell lines. The cells were seeded into 96-well cell culture plate at a density of 1 × 104/well and incubation for further 24 h. The cells were further maintained at 37 °C for 18 h under 5 % CO2 after Cy-met was added into each well with a concentration range from 0 to 20.0 µM. After another 18 h incubation, MTT solution (20 µL, 5.0 mg mL-1) was added into each well and incubated for another 4 h. The medium containing MTT was then carefully replaced by 150 µL of DMSO, which were added into each well. The plates were gently shaken for 15 min at room temperature before the absorbance ACS Paragon Plus Environment

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measurement. Six independent experiments were carried out for in vitro cytotoxicity analysis. The following formula was used to calculate the viability of cell growth: viability (%) = (mean absorbance of test wells - mean absorbance of medium control wells) / (mean absorbance of untreated wells - mean absorbance of medium control well) × 100%. 2.5 Confocal Fluorescence Imaging of Cy-Met in tumor Cells U87, MCF-7 and A549 cells, which are known to overexpress LAT1 transporter, are selected to evaluate cell affinity of Cy-Met.34 The cells were plated in laser scanning confocal microscope (LSCM) culture dishes with a density of 5×105 cells/well and allowed to adhere for 12 h at 37 °C in a humidified atmosphere containing 5 % CO2. When the space of culture dishes was took up 70 % - 80 %, the cells were washed with PBS buffer and then incubated with 10 µM Cy-Met or Cy-NH2 in DMSO/PBS buffer (1.0 : 99.0, v/v) for 0.5 h, 1 h, 2 h and 4 h at 37 °C, respectively. For mitochondria or nucleus staining, the cells were stained with Mito-Tracker Green (1.0 µM) or Hochest 33342 (1.0 µM) for 30 min and then washed with PBS buffer (2 mL × 3 times) to remove the free dye and Cy-Met or Cy-NH2. For blocking experiment, cells were incubated with BCH (1 mM) for 30 min at 37 °C in DMEM medium. After this period, cells were washed by PBS for three times and incubated with Cy-Met (10 µM) for another 2 h. Fluorescence images were detected on an FV1000 confocal fluorescence microscope (Olympus, Japan). The fluorescence signal of cells incubated with Cy-Met was collected at the NIR fluorescence channel (780 ± 30 nm, λex = 639 nm), green fluorescence channel (516 ± 30 nm, λex = 490 nm), blue fluorescence channel (461 ± 30 nm, λex = 340 nm) respectively. 2.6 In vivo dynamic bio-distribution of Cy-Met in normal mice Normal (ICR-SPF) mice were purchased from Qinglongshan Animal Center (Nanjing, China) for in vivo imaging investigation. All animal experiments were carried out in compliance with the Animal Management Rules of the Ministry of Health of People’s Republic of China (document no. 55, 2001) and the Guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University. The normal nude mice were intravenously injected with 100 µL of Cy-Met solution ACS Paragon Plus Environment


(30 µM, DMSO: PBS = 1.0: 99.0) and monitored by NIR fluorescence imaging system. After intravenously injection of Cy-Met, the NIR fluorescence images at different time intervals (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 60 h and 72 h) were obtained. In order to confirm the bio-distribution of Cy-Met in living mice, the mice were dissected and the main organs (heart, liver, spleen, lung, kidney and intestine) were collected for fluorescence imaging. The relative fluorescence intensity was determined by selecting the region of interest (ROI) and calculated using the ROI functions of Image-J software. The liver/muscle (L/M) ratio and intestine/muscle (I/M) ratio of the fluorescence intensity were defined as: (liver signal - background signal)/ (muscle signal - background signal) and (intestine signal - background signal)/ (muscle signal - background signal) respectively. 2.7 In vivo fluorescence imaging of tumor-bearing mice models To establish of the tumor models, ~5×106 of U87 (Human Glioblastoma cell) cells, H22 (hepatocellular carcinoma cell) or EAC (Ehrlich Ascites Tumor cell) were injected subcutaneously into the axillary fossa of the mice. After four weeks, the volume of the tumor size increased to around 100 mm3, and the mice were immobilized for in vivo NIR fluorescence imaging The tumor-bearing nude mice were intravenously injected with 100 µL of Cy-Met or Cy-NH2 solution (30 µM, DMSO: PBS = 1.0: 99.0) and monitored by NIR fluorescence imaging system. NIR fluorescence images were obtained at different time intervals, which were collected by a NIR fluorescence imaging system, equipped with a laser, a highly sensitive NIR CCD camera, an 800 nm (±12 nm) band pass filter and a condenser. Additionally, the mice were sacrificed and the main organs (heart, liver, spleen, lung, kidney, intestine and tumor) were collected for fluorescence imaging. The relative fluorescence intensity was determined by selecting the region of interest (ROI) and calculated using the ROI functions of Image J software. The tumor/normal tissue (T/N) ratio of the fluorescence intensity were defined as follows: T/N ratio (tumor/normal tissue ratio) = (tumor signal - background signal)/ (normal signal - background signal). For blocking experiment in vivo, the tumor-bearing nude mice were intravenously ACS Paragon Plus Environment

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injected with 100 µL of Cy-Met solution (30 µM, DMSO: PBS = 1.0: 99.0) after administration of 100 µL of methionine solution (3 mM, DMSO: PBS = 1.0: 99.0) for 30 min. NIR fluorescence images were obtained at different time intervals respectively. 2.8 Statistical analysis Significant differences were determined using the Student’s t-test where differences were considered significant (p < 0.05). All data are expressed as mean ± standard error of the mean. 3. Results and Discussion 3.1 Design and Synthesis of the probe An NIR fluorescence probe capable of efficient tumor-target imaging in vivo was well designed and synthesized following the scheme shown below with slight modifications (Scheme 1). This fluorescence probe was composed of a fluorescence reporting unit (an aminocyaine dye) and a tumor-targeting unit (L-Met). The fluorescence reporting unit was conjugated with tumor-targeting unit through a piperazin-polyamines linker to provide a target compound Cy-Met under basic conditions with a satisfactory yield of 63 %. All the structures of intermediate compound and Cy-Met were validated by 1H NMR, 13C NMR and ESI spectra (Figure S1-S9).



Scheme 1. Synthetic route of Cy-Met. a: anhydrous DMF, 85 °C, 4 h, 86 %; b: K2CO3, anhydrous CH3CN, 55 °C, 24 h, 70 %; c: 85% Hydrazine hydrate, ethyl alcohol absolute, 40 °C, 8 h, 44 %; d: PyBop, anhydrous DMF, DIEA, 2 h, 63 %.

3.2 Optical Properties of Cy-Met The absorbance and fluorescent spectra of compound 1, compound 2, Cy-NH2 and Cy-Met are shown in Figure 1. Cy-Met demonstrated the same maximum absorption peak as Cy-NH2 which located at 680 nm, whereas the double absorption peaks diminished to one absorption peak as Cy-NH2 conjugated with Met to form Cy-Met. The inserted photos in Figure 1A displayed the real color of the solutions of Cy-NH2 and Cy-Met. Meanwhile, the fluorescence peak of Cy-Met blue shifted from 805 nm to 790 nm in comparison with Cy-NH2. Moreover, the inserted NIR fluorescence images in Figure 1B indicated the strong fluorescence intensity of Cy-Met detected by NIR fluorescence imaging system.


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Figure 1. (A) Absorbance spectra of 1, 2, Cy-NH2 and Cy-Met. (10 µM, DMSO/PBS=1.0/99.0 v/v, 37 °C) Inset: real color photos of Cy-NH2 and Cy-Met. (B) Fluorescence spectra of compound 1, compound 2, Cy-NH2 and Cy-Met. Inset: corresponding fluorescence images of Cy-Met detected by NIR fluorescence imaging system.

3.3 Cytotoxicity Assay In order to evaluate the cell affinity of Cy-NH2 and Cy-Met under physiological conditions, the biocompatibility was performed on U87 cells and L02 cells firstly. The cytotoxicity of Cy-NH2 and Cy-Met were evaluated using conventional MTT assays (Figure 2). As indicated in Figure 2, even incubated with relatively high concentration of Cy-NH2 and Cy-Met (1~20 µM) for 18 h, the viability of cells can maintain above 80 %. Compared with Cy-NH2, Cy-Met showed lower cytotoxicity against U87 cells and L02 cells at the same concentration. Apparently, Cy-Met indicated improved biocompatibility ascribed to the introduction of L-Met, which is endogenic and hydrophilic amino acid. However, Cy-NH2 was a typical cyanine analogue which is characterized with hydrophobicity under physiological conditions. The results shown above validated the applicability of Cy-Met to act as a bio-probe for in vivo imaging.



Figure 2. In vitro cytotoxicity studies of Cy-NH2 and Cy-Met performed on the U87 (A) or L02 (B) cell by MTT assay. The viability of the cells without incubation with the probe is defined as 100 %. The results are shown as the mean ± standard deviation of six separate measurements.

3.4 Cell affinity evaluation of Cy-NH2 and Cy-Met As illustrated in Scheme 2, the LAT1 expressed at high levels in cancer cells. Cy-Met entered into cancer cells via the combination and transportation by LAT1.

Scheme 2. Schematic illustration of the transportation of Cy-Met into cancer cell mediated by LAT1 high-expressed on cancer cell.

LAT1 level in different cells were detected by SDS polyacrylamide gel


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electrophoresis (SDS-PAGE) method with GAPDH as the internal standard. As shown in Figure 3, the migration of LAT1 from L02 cell, U87 cell, A549 cell and MCF-7 cell were the same as that of the LAT1 marker. The level of LAT1 in tumor cells (A549, MCF-7, U87) and L02 cell were significantly higher than HUVEC group. The results proved the over-expression of LAT1 in U87, A549 and MCF-7 cell.

Figure 3. The expression analysis results of the LAT1 in different cell lines.

The cell affinity of Cy-NH2 and Cy-Met was further evaluated on LAT1 over-expressed cells (MCF-7 cells, A549 cells and U87 cells). As shown in Figure 4A, the green channel illustrated the visualization of the mitochondria by Mito Tracker Green and the red fluorescence corresponded to the emission signal from Cy-NH2 or Cy-Met. Obviously, all the three cells displayed strong red fluorescence 2 h after incubation with Cy-NH2 and Cy-Met. Additionally, the fluorescent intensity of Cy-Met is 1.5-fold higher than Cy-NH2, which could be attributed to methionine-mediated probe (Cy-Met) displayed higher binding ability to LAT1 high-expression cells compared with that of Cy-NH2. The semi-quantitative analysis of the fluorescence intensity at 2 h incubation with Cy-NH2 and Cy-Met manifested



the different cell affinity between Cy-Met and Cy-NH2 clearly (Figure 4B).

Figure 4. (A) Confocal fluorescence microscopy imaging of MCF-7, A549 and U87 cells incubated with Cy-NH2 (10 µM) and Cy-Met for 2 h. (B) The semi-quantitative fluorescent intensity analysis of Cy-NH2 and Cy-Met in the above three cell lines. The fluorescence imaging was collected at the Near-IR channel (780 ± 30 nm, λex = 639 nm). Scale bar = 10 µm.

Furthermore, the cells pre-incubated with BCH (Block group), a known inhibitor for System L, showed significantly weaker fluorescence than those incubated with only Cy-Met. The similar results of negative group (HUVEC cell) was obtained, which further validated the affinity of Cy-Met with LAT1 high expressed tumor cell (Figure 5).


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Figure 5. Confocal fluorescence microscopy imaging of U87 cells incubated with Cy-Met (10 µM), Cy-Met (10 µM) + BCH (1 mM) for 2 h; confocal fluorescence microscopy imaging of HUVEC cells incubated with Cy-Met (10 µM) for 2 h.

After confirming the cell affinity of Cy-Met, the time-dependent investigation was further carried out on U87 cells. The subcellular co-localization experiments were performed by co-staining U87 cells with Mito Tracker Green, Hochest and Cy-Met. As depicted in Figure 6A, as the incubation time extended, it showed a gradual increase of red fluorescence accompanied by a dramatic color change of merged channel from green to orange, indicating a substantial overlap of the fluorescence signal from Cy-Met and that from Mito Tracker Green. The fluorescence intensity reached a maximum at 2 h after incubation of the cell with Cy-Met with an increase of 2.3-fold compared to incubation at 0.5 h. As exhibited in Figure 6B, the semi-quantitative analysis of the fluorescence intensity indicated the apparent different fluorescence intensity at different incubation time (0.5 h, 1 h, 2 h and 4 h). As indicated in Figure 6C and 6D, fluorescence signal from Cy-Met showed substantial overlap with that of Mito Tracker Green. The fluorescent signal of linear regions of interest (ROI) showed tendency to synchronize with a Pearson’s co-localization coefficient of 0.88. The results described above revealed the capability



of Cy-Met to selective targeting mitochondria in tumor cells rather than other organelles. Additional, the time-dependent investigation of Cy-NH2 on U87 cells was carried out. As depicted in Figure S10, as the incubation time extended, the intensity of red fluorescence showed a gradual increase and reached a maximum at 2 h. The color of merged channel showed a dramatic color change from green to orange.

Figure 6. (A) Confocal fluorescence images of U87 cells incubated with Cy-Met (10 µM) at different time intervals (0.5 h, 1 h, 2 h and 4 h). All cells were stained with 1.0 µM Mito Tracker Green for mitochondrial staining, 1.0 µM Hochest for cell nucleus staining and 10 µM Cy-Met for fluorescence imaging. (B) The semi-quantitative fluorescent intensity analysis of Cy-Met at different time intervals. (C, D) Linear profile and scatter plot were used to characterize the overlap degree of red fluorescence from Cy-Met and green


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fluorescence from Mito Tracker Green. Scale bar = 20 µm.

3.5 In vivo dynamic bio-distribution of Cy-Met in normal mice Inspired by above promising results in living cell imaging, the capability of Cy-Met as an effective NIR fluorescence probe for tumor-targeting imaging in vivo was further investigated. To evaluate the dynamic bio-distribution of Cy-Met in vivo, 100 µL of Cy-Met solution (30 µM, DMSO: PBS = 1.0: 99.0) was intravenously injected into normal mice. As presented in Figure 7A, a strong fluorescence signal appeared in liver initiated at 1 h post-injection of Cy-Met and it lasted till 72 h post-injection. Attributed to the amino acid or peptide metabolism via the liver, Cy-Met was cleared mainly through the liver–intestine pathway. Furthermore, bio-distribution of Cy-Met in the main organs (heart, liver, spleen, lung, kidney and intestines) was studied by dissection at different time intervals (12 h, 24 h, 48 h and 72 h). As indicated in Figure 7B, Cy-Met accumulated mainly in liver the fluorescence in liver could be detected until 72 h. The Cy-Met accumulation in the intestines appeared after 24 h circulation. The liver/muscle (L/M) ratio and intestine/muscle (I/M) ratio of the Cy-Met at different time points were analyzed quantitatively (Figure 7C). The L/M ratio of Cy-Met always displays a high value and I/M ratio increased gradually until 72 h of post-injection. Histological images of intestines and liver at 48 h post-injection shown in Figure S11 indicated that Cy-Met did not induce obvious toxic side effects to animals.



Figure 7. (A) The dynamic distribution of Cy-Met in normal mice. (B) Bio-distribution of the Cy-Met in main organs (heart, liver, spleen, lung, kidney and intestines) detected by near-infrared (NIR) imaging system at different time points. (C) The liver/muscle (L/M) ratio and intestine/muscle (I/M) ratio of the Cy-Met at different time points.

3.6 In vivo tumor-target imaging in different animal models 3.6.1 In vivo tumor-target imaging in U87 tumor-bearing mice model


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Figure 8. (A) The in vivo NIR fluorescence images of Cy-Met in U87 tumor-bearing mice models at different time intervals; (B) Bio-distribution of the Cy-Met in main organs (heart, liver, spleen, lung, kidney, intestines and tumor) detected by NIR imaging system at 216 h of post-injection; (C) the tumor/normal tissue (T/N) ratios of Cy-Met at different time intervals.

In tumor cells, LAT1 is consistently expressed at high levels compared to normal tissue cells for transporting L-amino acids. Cy-Met was designed as an efficient probe that can target tumor precisely and longstanding. Herein, LAT1 over-expressed (U87, H22 and EAC) tumor-bearing mouse models were introduced to evaluate the capability of Cy-Met for tumor bio-targeting in vivo. As shown in Figure 8A, the fluorescence signal initially concentrated on liver in U87 tumor-bearing mouse within 2 h of post-injection of Cy-Met. At 6 h post-injection of Cy-Met, a distinct



fluorescence signal was observed in tumor and the fluorescent intensity increased with time, revealing tumor-targeting ability of probe. The fluorescence signal reached peak intensity at 120 h and maintained the high contrast ratio till 216 h, suggesting a long accumulation time of Cy-Met in tumor. Furthermore, bio-distribution of Cy-Met in the main organs and tumor was studied by dissection at 216 h of post-injection, indicating the principal accumulation in the tumor (Figure 8B). The semi-quantitative fluorescence analysis of tumor site was shown in Figure 8C. The T/N (tumor/normal tissue) ratio increased dramatically after 6 h of post-injection, and the peak value appeared at 120 h of post-injection. 3.6.2 In vivo tumor-target imaging in H22 tumor-bearing mice model The tumor-targeting capability was further investigated on H22 tumor-bearing mice model, as shown in Figure 9. As demonstrated in Figure 9A, the fluorescence signal initially concentrated in liver within 2 h of post-injection of Cy-Met. At 4 h post-injection of Cy-Met, a distinct fluorescence signal was observed in tumor and the fluorescent intensity increased along with the time. The fluorescence signal reached peak intensity at 72 h and maintained the high contrast till 120 h. The bio-distribution of the Cy-Met in the dissected organs showed that Cy-Met mostly accumulated in the tumor and liver at 72 h of post-injection (Figure 9B). The semi-quantitative fluorescence analysis of tumor site was carried out as well. As described in Figure 9C, the T/N (tumor/normal tissue) ratio initiated to increase after 4 h of post-injection, and the highest value appeared at 72 h of post-injection. However, the fluorescence signal was only observed in liver and intestine after injection of Cy-NH2. There was no fluorescence signal appear in the tumor site throughout the experiment (Figure S12). Blocking experiments were performed by the pre-injection of methionine. The results showed that the fluorescence signal appeared in tumor extended to 36 h post-injection of Cy-Met on H22 tumor-bearing model (Figure S13).


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Figure 9. (A) The dynamic distribution of Cy-Met in H22 tumor-bearing mice models; (B) Bio-distribution of the Cy-Met in main organs (heart, liver, spleen, lung, kidney, intestines and tumor) detected by near-infrared (NIR) imaging system at 72 h post-injection; (C) the tumor/normal tissue (T/N) ratio of Cy-Met at different time points.

3.6.3 In vivo tumor-target imaging in EAC tumor-bearing mice model Lastly, the tumor-targeting capability was further investigated on EAC tumor-bearing mice model (Figure 10). As displayed in Figure 10A, the fluorescence signal initially appeared on liver within 2 h of post-injection of Cy-Met. At 6 h post-injection of Cy-Met, an apparent fluorescence signal was observed in tumor and the fluorescent intensity increased as the time extended. The fluorescence signal reached peak intensity at 36 h and maintained the high contrast even at 72 h post-injection. Furthermore, the bio-distribution of the Cy-Met in main organs at 36 h ACS Paragon Plus Environment


of post-injection confirmed the main accumulation of the probe in the tumor and liver (Figure 10B). The semi-quantitative fluorescence analysis of tumor described in Figure 10C demonstrated the high T/N (tumor/normal tissue) ratio after 6 h of post-injection, and the highest value appeared at 36 h of post-injection. However, the fluorescence signal was only observed in liver and intestine after injection of Cy-NH2. There was no fluorescence signal appear in the tumor site throughout the experiment (Figure S14). Blocking experiments were performed by the pre-injection of methionine. The results showed that the fluorescence signal appeared in tumor extended to 48 h post-injection of Cy-Met on EAC tumor-bearing model (Figure S15).

Figure 10. (A) The dynamic distribution of Cy-Met in EAC tumor-bearing mice models; (B) Bio-distribution of the Cy-Met in main organs (heart, liver, spleen, lung, kidney, intestines


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and tumor) detected by near-infrared (NIR) imaging system at 36 h post-injection; (C) the tumor/normal tissue (T/N) ratio of Cy-Met at different time points.

In

previous

research,

Hazari

et

al,

synthesized

a

specific

SPECT-radiopharmaceutical for tumor imaging based on Met, which covalently conjugated two molecules of Met to diethylenetriaminepentaaceticacid (DTPA) and labeled with

99m

Tc.35 It revealed significant tumor uptake and good contrast in

tumor-bearing mice. More-important, it was competence in clinical diagnosis of metabolic tumors with the sensitivity, specificity, and positive predictive values found to be satisfying. However, the target-to-nontarget ratio was low and imaging period just maintained 25 h. In addition, SPECT had many limitations, such as high cost, relatively low resolution, narrow time window and radioactivity. NIR fluorescent probes combined with NIR imaging system, characterized with high resolution, noninvasive, sensitive, real-time way and low-cost, have been used as prominent imaging modality to imaging tumor both in vitro and in vivo. In this study, Met decorated NIR fluorescent probe was well developed for tumor imaging with a prominent high T/N ratio for easy detection in complicated biological system. In addition, Cy-Met is a low-cost symmetric dye whose synthesis and purification is relatively simple. Furthermore, Cy-Met overcomes the stability issue through a more robust C−C linkage as the linker chain. In this work, a Met decorated NIR fluorescent probe for prolonged tumor imaging was developed and notably validated, whose imaging time could maintain up to a few days with high tumor/normal contrast ratio. Nowadays, fluorescence-guided surgery (FGS) has emerged as an effective tumor therapy. As is known to all, surgery often takes a long time while some contrast agent could not meet the requirement. Fortunately, Cy-Met could provide excellent tumor contrast for monitoring the cancer surgery in human patients. Therefore, Cy-Met could also be served as a promising fluorescence probe for FGS. 4. Conclusions In summary, an effective IR-780-based fluorescent probe (Cy-Met) for prolonged tumor-targeting imaging was developed and successfully synthesized and validated. ACS Paragon Plus Environment


MTT assay and cell affinity study demonstrated that Cy-Met had low cytotoxicity and could be effectively up-taken by tumor cells. Meanwhile, Cy-Met was capable of selective targeting of mitochondria in cells. The in vivo tumor-targeting studies showed that Cy-Met had a high tumor-targeting ability in tumor-bearing mice with favorable sensitivity, high tumor/normal contrast ratio and long circulation time even up to a few days. The contribution of the affinity between Met and LAT-1 high-expressed in tumor cells facilitate the effective tumor-targeting ability of Cy-Met. Cy-Met is a promising NIR fluorescence probe with long-time tumor-targeting ability for tumor imaging, which can be exploited as an excellent tumor-targeting NIR fluorescence probe for clinic application in the future. Acknowledgments The authors are grateful to the Natural Science Foundation Committee of China (NSFC 81671803), National Key Research and Development Program (Grant No. 2017YFC0107700), the Outstanding Youth Foundation of Jiangsu Province (GX20171114003), and the Priority Academic Program Development of Jiangsu Higher Education Institutions for their financial support. Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI:XXXX. Syntheses, compound characterization data, confocal fluorescence images of Cy-NH2,

histological images, in vivo tumor-target imaging in H22/EAC

tumor-bearing mice models of Cy-NH2, in vivo blocking experiments in H22/EAC tumor-bearing mice models of Cy-NH2, quantum efficiency of fluorescence (PDF) Notes The authors declare no competing financial interest. References (1) Grundmann E, Höffken K. Journal of cancer research and clinical oncology. 2016:72-73. (2) Yang Z, Sweedler J V. Application of capillary electrophoresis for the early diagnosis of cancer. Anal Bional Chem. 2014, 406(17):4013-4031. ACS Paragon Plus Environment

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Table of Contents Graphic



Synthetic route of Cy-Met. a: anhydrous DMF, 85 °C, 4 h, 86 %; b: K2CO3, anhydrous CH3CN, 55 °C, 24 h, 70 %; c: 85% Hydrazine hydrate, ethyl alcohol absolute, 40 °C, 8 h, 44 %; d: PyBop, anhydrous DMF, DIEA, 2 h, 63 %. 110x93mm (300 x 300 DPI)


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(A) Absorbance spectra of 1, 2, Cy-NH2 and Cy-Met. (10 µM, DMSO/PBS=1.0/99.0 v/v, 37 °C) Inset: real color photos of Cy-NH2 and Cy-Met. (B) Fluorescence spectra of compound 1, compound 2, Cy-NH2 and CyMet. Inset: corresponding fluorescence images of Cy-Met detected by NIR fluorescence imaging system. 160x58mm (300 x 300 DPI)



In vitro cytotoxicity studies of Cy-NH2 and Cy-Met performed on the U87 (A) or L02 (B) cell by MTT assay. The viability of the cells without incubation with the probe is defined as 100 %. The results are shown as the mean ± standard deviation of six separate measurements. 160x61mm (300 x 300 DPI)


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Schematic illustration of the transportation of Cy-Met into cancer cell mediated by LAT1 high-expressed on cancer cell. 101x115mm (300 x 300 DPI)



The expression analysis results of the LAT1 in different cell lines. 45x47mm (300 x 300 DPI)


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(A) Confocal fluorescence microscopy imaging of MCF-7, A549 and U87 cells incubated with Cy-NH2 (10 µM) and Cy-Met for 2 h. (B) The semi-quantitative fluorescent intensity analysis of Cy-NH2 and Cy-Met in the above three cell lines. The fluorescence imaging was collected at the Near-IR channel (780 ± 30 nm, λex = 639 nm). Scale bar = 10 µm. 69x61mm (300 x 300 DPI)



Confocal fluorescence microscopy imaging of U87 cells incubated with Cy-Met (10 µM), Cy-Met (10 µM) + BCH (1 mM) for 2 h; confocal fluorescence microscopy imaging of HUVEC cells incubated with Cy-Met (10 µM) for 2 h. 45x32mm (300 x 300 DPI)


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(A) Confocal fluorescence images of U87 cells incubated with Cy-Met (10 µM) at different time intervals (0.5 h, 1 h, 2 h and 4 h). All cells were stained with 1.0 µM Mito Tracker Green for mitochondrial staining, 1.0 µM Hochest for cell nucleus staining and 10 µM Cy-Met for fluorescence imaging. (B) The semi-quantitative fluorescent intensity analysis of Cy-Met at different time intervals. (C, D) Linear profile and scatter plot were used to characterize the overlap degree of red fluorescence from Cy-Met and green fluorescence from Mito Tracker Green. Scale bar = 20 µm. 93x102mm (300 x 300 DPI)



(A) The dynamic distribution of Cy-Met in normal mice. (B) Bio-distribution of the Cy-Met in main organs (heart, liver, spleen, lung, kidney and intestines) detected by near-infrared (NIR) imaging system at different time points. (C) The liver/muscle (L/M) ratio and intestine/muscle (I/M) ratio of the Cy-Met at different time points. 93x116mm (300 x 300 DPI)


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(A) The in vivo NIR fluorescence images of Cy-Met in U87 tumor-bearing mice models at different time intervals; (B) Bio-distribution of the Cy-Met in main organs (heart, liver, spleen, lung, kidney, intestines and tumor) detected by NIR imaging system at 216 h of post-injection; (C) the tumor/normal tissue (T/N) ratios of Cy-Met at different time intervals. 93x100mm (300 x 300 DPI)



(A) The dynamic distribution of Cy-Met in H22 tumor-bearing mice models; (B) Bio-distribution of the CyMet in main organs (heart, liver, spleen, lung, kidney, intestines and tumor) detected by near-infrared (NIR) imaging system at 72 h post-injection; (C) the tumor/normal tissue (T/N) ratio of Cy-Met at different time points. 49x52mm (300 x 300 DPI)


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(A) The dynamic distribution of Cy-Met in EAC tumor-bearing mice models; (B) Bio-distribution of the CyMet in main organs (heart, liver, spleen, lung, kidney, intestines and tumor) detected by near-infrared (NIR) imaging system at 36 h post-injection; (C) the tumor/normal tissue (T/N) ratio of Cy-Met at different time points. 46x45mm (300 x 300 DPI)


Methionine-Decorated Near Infrared Fluorescent Probe for Prolonged

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